Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Li Ka Shing Institute of Virology at University of Alberta, Edmonton, Alberta, Canada.
PLoS Negl Trop Dis. 2020 Jun 4;14(6):e0008283. doi: 10.1371/journal.pntd.0008283. eCollection 2020 Jun.
The Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity.
METHODOLOGY/PRINCIPLE FINDINGS: Recombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2'-deoxy-2'-fluoro-CTP (FdC) and 2'-deoxy-2'-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions.
CONCLUSIONS/SIGNIFICANCE: The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.
克里米亚-刚果出血热病毒（CCHFV）是一种具有负义 RNA 基因组的病毒，能够引起严重的人类疾病。世界卫生组织（WHO）已将 CCHFV 列为具有优先病原体地位的病毒，并迫切需要加强研究活动以开发有效的对策。在这里，我们采用了针对病毒 RNA 依赖性 RNA 聚合酶（RdRp）的生化方法。CCHFV RdRp 活性是一种多功能 L 蛋白的一部分，该蛋白异常大，分子量约为 450kDa。CCHFV L 蛋白还包含具有去泛素化（DUB）活性的卵巢肿瘤（OTU）结构域，该结构域被证明会干扰先天免疫反应和病毒复制。我们报告了全长 CCHFV L 蛋白的表达、表征和抑制，并研究了 RNA 合成和 DUB 活性。
方法/原理发现：使用亲和层析法从昆虫细胞中表达和纯化了重组全长 CCHFV L 蛋白。在二价金属离子存在的情况下，通过在延伸过程中使用模型引物/模板监测 RdRp 活性。我们观察到 14 个核苷酸的全长 RNA 产物，以及当从反应混合物中省略某些核苷酸时，预期的较短产物。假定活性位点的 D2517N 突变使酶失活。用广谱抗病毒药物利巴韦林和法匹拉韦研究 RNA 合成的抑制作用，这些药物模拟核苷酸底物。这些化合物的三磷酸形式类似于 ATP 或 GTP；然而，与抑制剂相比，ATP 或 GTP 的掺入明显更有利。我们还研究了真正的核苷酸类似物 2'-脱氧-2'-氟-CMP（FdC）和 2'-脱氧-2'-氨基-CMP 的作用，并证明由于掺入率更高，抑制作用增强。我们进一步表明，全长 CCHFV L 蛋白和分离的 OTU 结构域可切割赖氨酸 48-和赖氨酸 63-连接的多聚泛素链。此外，泛素类似物 CC.4 以相似的程度抑制全长 L 蛋白和分离的 DUB 结构域的 CCHFV 相关 DUB 活性。DUB 活性的抑制不影响 RNA 合成的延伸，而 RNA 合成的抑制不影响 DUB 活性。在这些条件下，两个结构域在功能上是独立的。
结论/意义：高生物安全措施的要求阻碍了具有传染性 CCHFV 的药物发现和开发工作。全长 CCHFV L 蛋白的可用性为此类研究提供了重要工具。现在可以进行高通量筛选（HTS）活动。相同的酶制剂可用于鉴定新型聚合酶和 DUB 抑制剂。
